Modern Mass Spectrometry and Coupling Techniques

Modern Mass Spectrometry and Coupling Techniques Rob Nieckarz Office: HCI D325 nieckarz@org.chem.ethz.ch Special Thanks to Dr. Richard Smith at the University of Waterloo

MS + Gas Chromatography Liquid Chromatography Capillary Electrophoresis Spectroscopy MS = Added informaoon and greater confidence in our analysis

NOTE: Mass Separation and the Lorentz Force All mass analyzers function on the basis of the Lorentz Force equation which describes the force exerted on a charged particle in an electromagnetic field. The particle will experience a force due to the electric field (qe), and due to the magnetic field (qv x B). Combined they give the Lorentz force equation: F = q(e + v x B) F is the force (in newtons) q is the electric charge of the particle (in coulombs) = ze E is the electric field (in volts per meter) B is the magnetic field (in webers per square meter, or equivalently, teslas) v is the instantaneous velocity of the particle (in m/s) q = ze therefore, F = zee+zevb 2

Mass resolution: Definition Valley Peak width at half height By courtesy of Spektrum Akademischer Verlag Mass Spectrometry September 2004 4

Mass Resolution The FWHM definition is easier to apply (only need one peak), but gives a resolution about twice that of the 10% valley definition Resolution for sector instruments is usually given as the 10% valley figure. High resolution has some obvious advantages: -It allows one to resolve ions that are isobaric -The narrower a peak, the easier it is to measure its position accurately 15

Why do we need high resolution? To resolve single, adjacent peaks of high molecular weight compounds To enhance specificity To determine the elemental composition of a compound: Example: Three different molecules may have the nominal mass 28: CO = 27.9949 ΔM = 0.0112 M/ΔM = 2800 N 2 = 28.0061 ΔM = 0.0252 M/ΔM = 1120 C 2 H 4 = 28.0313 Mass Spectrometry September 2004 5

Mass resolution: peak shape By courtesy of Spektrum Akademischer Verlag Mass Spectrometry September 2004 6

Mass Resolution Low resolution: <2,000. Suitable only for nominal mass measurement. Medium resolution: 2,000-20,000. Suitable for accurate mass measurement. Resolve isotope clusters of high charge states. High resolution: >20,000. Better than medium resolution. You can never have too much resolution! In practice, there is a trade-off between resolution and sensitivity. The ions are not coming from a point source: they exit the source through a slit of finite dimensions, and cannot be perfectly focussed. Slits and lens help to compensate for this by cutting out ions from the centre of the beam and focussing. To get very high resolution, the slits have to be narrowed, which means that a lot of ions are lost. 16

Types of Mass Analyzers: Time of flight mass spectrometers (Tof) Fourier transform ion cyclotron resonance (FTICR) Orbitrap Linear quadrupoles (Q - mass filters) Three dimensional quadrupoles (ion traps - IT) Linear iontraps (2D) Sector instruments Tandem instruments

Time of Flight Mass Analyzer Fast, simple, high mass accuracy and resoluoon

Time of Flight (Tof) Principle: Ions of different mass (accelerated by the same field, V) have different velocities and thus flight times. The larger the mass the slower the ion: K.E. = zev = mv²/2 Ion formation: Ions are introduced to the Tof in pulses (e.g. MALDI or orthogonal extraction from a continuous beam such as ESI) Ion detected by analogue or time to digital converter (GHz ADC or TDC) Linear Tof (high mass range but low mass resolution) Reflectron Tof (lower mass range but high mass resolution) 53

Mass Separation: Time of Flight (Tof) MS acceleration region (drift region) 54

Basic Principles Since the initial kinetic energy of the ions is given by: zev = mv²/2 velocity: v = (2zeV/m) 1/2 time of flight: t = L/v = L[m/(2zeV)] 1/2 m/z = 2eVt 2 /L 2 Example: For C 6 H +. 5 and C 7 H +. 7, (m/z 77 and 91), accelerated at 10kV, what are the velocities of these 2 ions and how long would it take them to traverse a 2m flight tube? using eq n (ii) v 77 = (2x1x1.6022x10-19 x10,000/m) 1/2 m(kg) = 0.077/6.022x10 23 = 1.279x10-25 v 77 = 158,306m/s = 9498 km/h or 12.63μs similarly for v 91 v 91 = 145,621m/s = 8737 km/h or 13.73μs (i) (ii) (iii) (iv) V is the extraction pulse potential (V) L is the length of field free drift zone (m) t is the measured time-of-flight of the ion (s) e = 1.6022x10-19 C 55

Example cont From eq (iii), difference in flight time: t A /t B = (m A /m B ) 1/2 Consequently, this square root relationship causes Δt for a given Δm/z to decrease with increasing m/z For example: Δt/amu is calculated to be 114ns at m/z 20 to be 36ns at m/z 200 to be 11ns at m/z 2000 Tof mass analyzer depends on the ability to accurately measure these short time intervals to make it a useful MS 56

Linear Tof Transmittance as high as 90% Ions introduced into the flight tube have a temporal and kinetic energy distribution which yields relatively poor mass resolution. Kinetic energy spread can be reduced by employing Delayed Ion Extraction Principle of Delayed Ion Extraction: Ions are formed during a short pulse of a few nanoseconds The acceleration (extraction) field is only applied after a delay of some hundreds of nanoseconds: At the beginning of the extraction ions with high initial velocities have traveled further than slower ones. Therefore after the second extraction pulse they do not experience the full acceleration potential. Thus the initially faster ions will be accelerated less than the initially 57 slower ions.

Reflectron Tof Same m/z but different kinetic energy In a reflectron Tof, the ions traverse the drift tube and penetrate into an electric field (ion mirror) where their direction is reversed. Faster ions (with higher kinetic energy) penetrate farther into the electric field than slower ions (with lower kinetic energy). Thus faster ions have a longer flight path and therefore need approximately the same flight time as the slower ions which have a shorter flight path. 58

Tof: Advantages and Disadvantages Good mass accuracy reflectron ~ 5-10ppm limited with quadrupole MS, poor with ion traps and linear Tof High mass resolution reflectron ~5,000 to 20,000 Quadrupole MS, ion traps and linear Tof operate closer to unit mass resolution at m/z ~ 10 3 High mass range linear >10 5 Da, reflectron <10 4 Da Ion traps and quadrupoles are limited to ~6,000 Da Acceptable linearity for linear and reflectron Tof not as good as quadrupole MS, but similar to ion traps Very good scan-to-scan reproducibility for linear and reflectron Tof as good as quadrupole MS Very fast aquisition time (up to khz) 59

Fourier Transform Ion Cyclotron Resonance Mass Analyzer UlOmate in mass accuracy and resoluoon Expensive, difficult to operate

Basic Construction: FTICR MS a cell where ions are trapped by intense, constant magnetic field and applied voltage The cell accepts ions in a pulsed mode from the continuous ion beam Detection of the ions is based on the FT deconvolution of the image current the circulating ions induce in a pair of detector plates after excitation with a resonant R f pulse. 61

Ion Trapping and FTICR MS ions enter the cell (or are created internally) and they begin their cyclotron motion, orbiting around the centre of the magnetic field since the magnetic field is quite high (typical minimum of 4.7T, but this is increasing) the ions are trapped in the radial (x,y) direction. Resolving power and scan speed increase linearly with B 63

Ion Trapping and FTICR MS by applying small, equal potentials to the two end or trapping electrodes, the ions are confined in the z or axial direction. ions can be confined for very long periods of time such that ion/molecule reactions or even slow unimolecular dissociation processes can be observed and monitored. 64

Cyclotron frequency Trapping oscillakon frequency Magnetron frequency m T

FTICR MS Detection In FT detection, all ions, regardless of their mass are detected at the same time. Ions before excitation. They have their natural cyclotron radius within the magnetic field. Once ions are trapped inside the ICR cell they are excited by a fast sweep of all the R f frequencies, exciting the ions to cyclotron motion with a larger radius. 65

FTICR MS - Detection When a packet of ions (+ve) approaches an electrode, electrons are attracted from ground and accumulate in that electrode causing a temporary current. As the ions continue to orbit, the electrons accumulate in the other electrode. The flow of electrons in the external circuit represents an image current. The amplitude of the current is proportional to the number of ions in the packet. 67

FTICR - Detection R f Excitation Time Detected time domain image current Time Fourier Transform Resulting mass domain Spectrum m/z the frequency of the image current oscillation is the same as the frequency of the ion s cyclotron motion which is related to mass. A small AC voltage is created across a resistor and is amplified and detected. using FT techniques all ion packets, each containing ions of the same mass, are detected. The decay of the image current (as the excited cyclotron orbit radius decays) is detected in time and transformed into a frequency domain signal by a 68 Fourier transform.

How fast are the ions moving? d = 0.05 m Frequency = 1 x 10 6 s - 1 Time for one revoluoon = 1 x 10-6 s Distance travelled = πd = 0.157 m Speed = 0.157 m / 1x 10-6 s = 157 000 m/s = 9 424 km/hr

FTICRMS Very high resolution is possible. The current record is 8x10 8, and routine values are 100,000 or so. Long trapping times are possible, allowing for ion-molecule reactions. Good sensitivity. Like the ion trap, the FTICR cell works well with pulsed sources. MS n capability However, expensive because of the cost of superconducting magnets and the very high vacuum requirements. Difficult to operate 69

Orbitrap Mass Analyzer The only new mass spectrometer concept to be developed in the last 30 years The only commercial instrument that can come close to the performance of an FTICR

Orbitrap Summary - High performance mass analyzer - Resolu:on up to 200,000 - Mass Range up to 50,000 - High mass accuracy (1-2 ppm) - Non- destruc:ve ion analyzer - MS n possible - CID und H/D exchange possible

Quadrupole Mass Analyzer Cheapest instrument Fast acquisioon Omes

Linear Quadrupoles (2D - mass filters) 20

Linear Quadrupoles (2D - mass filters) Four hyperbolic rods (cheap version: circular rods) compromise! Opposite pairs of rods are connected electrically but are of opposite polarity Each pair of rods has a DC (U) + AC (V 0 cosωt) R f voltage applied: 1 pair of rods: -(U + V 0 cosωt) and the opposite pair: +(U + V 0 cosωt) where, ω = radial frequency = 2πf During a mass scan, the DC and AC voltages are ramped but the ratio of DC/AC (ie U/V 0 )is kept constant For a given DC and AC amplitude, only ions with a given m/z (or m/z range) have stable oscillations and are transmitted and can be detected 21

Quadrupole (end view) Hyperbolic Round Equipotential Field Lines 22

Linear Q: Equations of Motion From the electrical part of the Lorentz equation, we can derive the equation of motion (x and y directions) for a particle in a combination of DC and AC R f fields the Mathieu equation: 2 du + ( au 2qucos 2ξ ) u= 0 2 dξ u represents the x or y transverse displacement. We do not consider displacement in the z direction because the electric field is 0 along the asymptotes of the hyperbolic rods. The 2 parameters characteristc of the field (a and q) are given by: a a 8zeU and = q x = y = 2 2 x y 2 2 mr0 ϖ mr0 ϖ q = 4zeV 25

Linear Q: Equations of Motion Where the variable ξ is the time in radians of the applied field = ωt/2 U is the DC voltage and V is the AC R f voltage of frequency ω r 0 is the radius of the instrument aperture Plotting a against q gives the Mathieu stability diagram of the linear quadrupole field - a/q = 2U/V Typical values are: - U = DC voltage (~200-1000V) - V = AC voltage (~1000-6000V, 1-2MHz), - m = mass of ion, e = electonic charge, z = # of charges on ion -2r 0 = distance between the rods - 1-2 cm 26

Stability Diagram 27

aq Space Note: Both +ve and ve abscissa with a values ranging up to 10 and q values ranging up to 20 In practice we only operate in the +ve area of region I Why? Because in order to have a and q values >1 we would require VERY high DC and AC voltages which is not practical 28

Stability Diagram a 0.3 0.2 0.1 Y unstable.. Operating lines a/q constant........ L 1 L 1 = L 2 L 2 L 1, only 1 ion has a stable trajectory all others ions are lost therefore adjacent ions are resolved from each other X unstable L 2, 3 ions have a stable trajectory at the same time therefore these 3 ions would not be resolved from each other In practice, the ratio of a/q is changed by changing the DC voltage 0.4 X and Y Stable 0.8 q What would happen if no DC voltage is applied? 29

a Conceptualizing a Q scan 0.3 stable region of m 1 Operating or scan line 0.2 stable region of m 2 m 1 < m 2 < m 3 stable region of m 3 0.1 0.4 0.8 q 31

Mass Range and Resolution Depends on 5 parameters: Rod length (L) 50 to 250mm Rod diameter (r) 6 to 15mm aligned to μm accuracy Maximum supply voltage (V m ) AC (R f ) fequency (f) Ion injection energy (V z ) - ~5 volts From the theory of quadrupole operation the following relationship can be derived: M max = 7x10 6 V m /f 2 r 2 Consequently, as r and f increase, M max decreases and as r and f decrease, M max increases 32

Mass Range and Resolution The resolution limit of a quadrupole is governed by the number of cycles of the R f field to which the ions are exposed: 2 M/ΔM = 0.05 fl m/2ev z Consequently, as both f and L increase so does resolution. If L in increased then f can be decreased and vice versa Scanning speeds as high as 6,000 amu/sec and mass resolution of 10,000 is attainable 33

Linear Q Advantages: Small and light weight ~20 cm long Inexpensive Simple to operate complete computer control Low accelerating voltage handles high source pressures better Full scan mass spectra and selected ion monitoring (SIM) for quantitation Disadvantages: Unit mass resolution only and limited mass range High mass discrimination Rod contamination causes further imperfections in the quadrupole field compromises resolution and sensitivity 34

Linear Q Other applications: QQQ for MS/MS Hybrid instruments eg BEQQ and QqTof Ion lenses (hexapoles and octapoles) Collision chambers for MS/MS ie QQQ and BEQQ etc Prefilter before mass resolving rods to reduce contamination 35

Quadrupole 3D Ion Trap (QIT) Ion trap consists of three electrodes: Cap Cap Ring r 0 Cap ring electrode (hyperbolic shape) 2 hyperbolic electrodes - end caps Orifice for ion injection Orifice for ion ejection Pulsed introduction of ions 36

QIT (properties) Ion trap volume very small (7mm i.d.) High sensitivity (10-18 mol) (scan mode) High mass range : 6,000 Higher mass resolution than Q ~x2-3 High dynamic range: 10 6 depending on space charging MS n capabilities Low mass cut-off is a disadvantage Helium is introduced intentionally into the ion trap (10 3 mbar) Needed as a buffer to absorb kinetic energy of incoming ions without chemical interaction so they can feel the effect of the trapping field - dampening (cooling) of oscillations collision partner for MS/MS and MS n Ions are concentrated in center of ion trap Better resolution and better sensitivity than Q 38

QIT (ion motion) Between the three electrode a quadrupole field exists, which forces the ions to the center of the trap The farther the ion is removed from center of trap the stronger is the exerted electric force The ions oscillate within the trap, but with a rather complex sinusoidal motion The ion motion can be described by Mathieu s differential equations 39

Quadrupole 3D Ion Trap (QIT) For the QIT, the electric field has to be considered in 3 dimensions. The electric field can be descibed by the expression: Φ x,y,z = Φ0(r 2-2z 2 ) r 0 2 The Mathieu equation still applies: 2 du + ( au 2qucos 2ξ) u= 0 2 dξ The equations of ion motion in the r and z direction are: d²z/dt² - (4e/mr 0 ²) [(U - V cos2ωt)z = 0 d²r/dt² + (2e/mr 0 ²) [(U - V cos2ωt)r = 0 40

Quadrupole 3D Ion Trap (QIT) Solving these Mathieu type differential equations yields the parameters a z and q z a z = -2a r = 16zeU m(r 0 ²+ 2z 02 )ω² and q z = -2q r = -8zeV m(r 0 ²+ 2z 02 )ω² Where ω = 2πf, f = fundamental R f frequency of the trap (~1MHz) 41

QIT (Ion stability diagram) Endcap Ring Electrode q = 0.908 q < 0.908 Ring Electrode Endcap 42 courtesy of Spektrum Akademischer Verlag

QIT (stability diagram) Ions are only stable both in r and z direction for certain defined values of a and q Ions oscillate with so called secular frequency, f, which differs from the frequency of applied R f field because of inertia (in addition oscillations of higher order) Ions of different m/z are simultaneously trapped, V determines low mass cut-off at q z = 0.908, which increases with V 43

QIT (mass selective ion stability scan) Mass scan is possible by increasing the amplitude of the voltage on the ring electrode (U = 0, a z = 0 ie no DC voltage) Scan line: While scanning along this line (a=0) ions become increasingly non stable and exit the stability diagram at q z = 0.908. Trajectory of these ions in z- direction. Ions exit from trap through holes in end cap. Linear scan function 44

Space Charging ~ 300 Ions ~ 1500 Ions ~ 3000 Ions ~ 6000 Ions 524.3 524.4 524.5 524.8 100 100 100 100 Relative Abundance 80 60 40 525.3 20 20 20 20 526.5 526.3 526.3 527.5 527.5 0 0 0 0 522 530 522 530 522 530 522 m/z Good resolution and mass accuracy 80 60 40 525.4 80 60 40 525.5 80 60 40 525.7 526.7 530 Poor resolution and mass accuracy 47 Courtesy of Agilent

QIT (space charge) With increasing number of ions trapped the space charge increases Space charge distorts the electric field Deterioration of resolution, sensitivity and mass accuracy Solution: Pre-scan or measure in real time to control the number of ions (or more correctly, the number of charges) in the trap (a maximum of ~10 3-10 4 ) 48

Linear (2D) traps Similar idea to 3D traps with a new 2D geometry R f only quads with DC voltage end electrodes Larger size than 3D IT higher ion capacity (~x50) therefore fewer space charge problems More than one design for this type of trapping instrument Hybrids such as QQQ where Q3 can also be used as a linear trap and LT-FTICR 49

Trapping Forces in a Linear Ion Trap Radial Trapping RF Voltage Axial Trapping DC Voltage Axial Trapping Courtesy of Sciex Radial Trapping RF Voltage Resonance Excitation Exit Lens 50

Linear Ion Trap vs 3D Trap No low mass cut-off Trapping Efficiency: >10 Detection Efficiency: doubled Overall Efficiency: >10 Ion Capacity (Spectral): >20 Scan Rate (amu/sec): 4x Highly Efficient MS n : 5x over 3D IT 52

Electric and MagneOc Sector Mass Analyzer The original commercial MS systems High resoluoon, but comes at the cost of sensiovity detector!

Mass Separation: Magnetic Fields m/z = eb 2 r 2 /2V Therefore specific values of V and B allow ions unique in m/z to pass to the detector. Variations in V or B will cause ions to collide with the walls of the flight tube therefore at any unique value of V or B only one specific ion will be passed to the detector. In practice only B scans are preferrred when generating full scan data over a large (>50Da) mass range One exception to this is when high resolution, accurate mass measurements are made where V acc scanning is preferred as voltages can be controlled and measured much more accuartely than can B 5

Deflection of ions of different masses in a constant magnetic field This is how Aston s original mass spectrograph operated! In modern instruments, the magnetic field is scanned to bring ions of different m/z ratios successively to the detector 7

Directional (angular) focusing of a magnetic field Divergent ions of the same m/z will be brought into focus by a magnetic field 8

Mass Separation: Magnetic Fields One significant drawback with employing B scans is that the initially accelerated ions have a kinetic energy spread which exhibits itself as increased peak width ie low resolution. To overcome this problem an electric sector (ESA) is combined with the magnetic sector to produce what is called a double focusing instrument. 9

New Developments in Magnetic Sector Instruments Large, high field magnets Mass range up to 10,000 Da at full accelerating potential (10 kv) for analysis of large biopolymers Example: bovine insulin (MW 5734) Laminated magnets To reduce magnetic hysteresis Total cycle time < 1 sec, fast scanning 19

Petroleum Hydrocarbons Analysis of diesel fuel: typically by GC or 2D GC- (TOF)MS C15 Analysis of diesel fuel sample 1:1000 in DCM, 60m DB5 column C9 C21 what other compounds can be detected with ultra high resolukon mass spectrometry? 8

Diesel 1:1000 in MeOH + 0.2% formic acid Zoom 9

Analysis of diesel fuel sample Zoom 10

Analysis of diesel fuel sample Zoom 11

Analysis of diesel fuel sample C 18 H 22 K +1 (0.32 ppm) C 17 H 18 O 2 Na +1 (0.00 ppm) C 14 H 22 O 4 Na +1 (0.11 ppm) C 15 H 26 O 2 K +1 (0.14 ppm) C 18 H 22 ONa +1 (0.40 ppm) C 15 H 26 O 3 Na +1 (0.28 ppm) C 13 H 18 O 5 Na +1 (0.18 ppm) C 16 H 30 O 2 Na +1 (0.00 ppm) C 13 H 18 O 4 K +1 (0.11 ppm) C 17 H 34 ONa +1 (0.03 ppm) 12

C 17 H 18 O 2 Na +1 (0.00 ppm) Analysis of diesel fuel sample 110,000 FWHM 2048K data points 29,000 FWHM 512K data points 7,000 FWHM 128K data points 1,500 FWHM 32K data points 13

Analysis of diesel fuel sample 110,000 FWHM 2048K data points 29,000 FWHM 512K data points 7,000 FWHM 128K data points 1,500 FWHM 32K data points 14

Analysis of diesel fuel sample 110,000 FWHM 2048K data points 29,000 FWHM 512K data points 7,000 FWHM 128K data points 1,500 FWHM 32K data points 15

Analysis of diesel fuel sample 110,000 FWHM 2048K data points 29,000 FWHM 512K data points 7,000 FWHM 128K data points 1,500 FWHM 32K data points 16